Abstract: Brain activity is driven in large part by neurotransmitter secretion, which is derived from a small pool of recycling synaptic vesicles (SVs). Following exocytosis, membrane and protein components of synaptic vesicles are incorporated into the plasma membrane and must be retrieved by endocytosis to maintain continued synaptic function. Subtle changes in SV endocytosis can lead to severe defects in brain function. The long-term goal of this project is to determine the molecular mechanisms governing SV endocytosis. Here, we will investigate Endophilin, a conserved protein required for SV endocytosis. In preliminary studies, we showed that Endophilin promotes endocytosis by bending membranes whereas its function as a molecular scaffold is not required for endocytosis. We showed that the majority of Endophilin at nerve terminals is bound to SVs, not at endocytic sites. We further showed that the rate of Endophilin unbinding from SVs is regulated by exocytosis. Based on these preliminary results, we propose three Aims. In Aim 1, we develop real-time assays for Endophilin's membrane association, oligomerization, and membrane bending. We will use these assay to determine if the rate of membrane bending is sufficiently fast to account for Endophilin's function in endocytosis. In Aim 2, we will determine how Endophilin is targeted to SVs. We hypothesize that inactive Endophilin monomers bind to the SV protein RAB-3 and thereby recruit Endophilin to the SV pool. We will use biochemical, genetic, and imaging approaches to test this idea. We will also determine if phosphatidylinositol 4,5-bisphosphate (PIP2) stimulates Endophilin oligomerization at endocytic sites. In Aim 3, we will determine how Synaptojanin's PIP2 phosphatase activity is coupled to endocytosis. Specifically, we will define the molecular mechanisms that allow Synaptojanin to sense membrane curvature generated by Endophilin. These studies will provide significant new insights into the mechanisms regulating SV endocytosis and how BAR domain proteins function generally.